Surgical device for skin therapy or testing

ABSTRACT

A device, and method of making the device, capable of therapeutic treatment and/or for in vitro testing of human skin. The device may be used on skin wounds for burned, injured, or diseased skin, and provides structures and functions as in normal uninjured skin, such as barrier function, which is a definitive property of normal skin. The device contains cultured dermal and epidermal cells on a biocompatible, biodegradable reticulated matrix. All or part of the cells may be autologous, from the recipient of the cultured skin device, which advantageously eliminates concerns of tissue compatibility. The cells may also be modified genetically to provide one or more factors to facilitate healing of the engrafted skin replacement, such as an angiogenic factor to stimulate growth of blood vessels. The inventive device is easy to handle and manipulate for surgical transplant, can be made into large sheets to minimize the number of grafts required to cover a large surface area to be treated, and can be produced within the time frame to treat a burned individual requiring a skin graft.

This application is a continuation of co-pending U.S. patent applicationSer. No. 12/818,302 filed Jun. 18, 2010, which is a continuation of U.S.Patent Application Ser. No. 10/092,237 filed Mar. 6, 2002 now U.S. Pat.No. 7,741,116, each of which is expressly incorporated by referenceherein in its entirety.

This invention was made with government support under Grant Nos.GM050509 and FD-R-000672 awarded by the National Institutes of Healthand the Food and Drug Administration, respectively.

FIELD OF THE INVENTION

The invention is directed generally to a surgical device for therapeutictreatment of skin wounds in a patient or testing of skin anatomy orphysiology, and a method to prepare the device.

BACKGROUND

Skin is one of the largest organs in the body and covers substantiallythe entire outer surface of the body. Skin is composed of two mainlayers: the surface epithelium or epidermis, which containskeratinocytes

-   as one type of epidermal cells, and the subjacent connective tissue    layer or dermis, which contains fibroblasts as one type of dermal    cells. The functions of skin include protecting an organism from    injury and dessication by serving as a barrier to infection,    perceiving or detecting environmental stimuli, excreting various    substances, regulating body temperature, and helping to maintain    water balance. Because of its quantitative and qualitative    importance, substantially intact and healthy skin is important, not    only for the well being of an organism but for its very survival.

The health and integrity of skin may be compromised by congenital oracquired pathologic conditions, either acute or chronic, for whichnormal skin regeneration and repair processes may be inadequate. Theseconditions include burns, wounds, ulcers, infections, diseases and/orcongenital abnormalities. Patients who are burned over a large surfacearea often require immediate and extensive skin replacement. Lesslife-threatening but chronic skin conditions, as occur in venous stasis,diabetic or decubitus ulcers as three examples, may progress to moresevere conditions if left untreated, particularly because patients withthese conditions have an underlying pathology. Reducing the morbidityand mortality in such patients depends upon timely and effectiverestoration of the structure and function of skin.

Skin substitutes derived either ex vivo or in vitro may be used to treatthese or other conditions. Desirable properties of skin substitutes areready availability, a minimum requirement for donor skin, relativesimplicity to produce, and cost-effectiveness of fabrication and use.Several approaches to fabrication of skin substitutes which satisfy someor all of these requirements have been attempted, with varying degreesof success. However, no skin substitute has yet regenerated all of thestructures and functions of skin. Rather, all are subsets of uninjuredskin. Only a transplant of full thickness skin restores virtually allthe structures and functions of normal uninjured skin, but furthermore,scars during healing.

Materials have been manufactured for therapeutic use in skin repair.These materials contain different components replacing or substitutingthe structures and functions of the dermis and/or epidermis. Examples ofthese materials include EpiCel™, which lacks a dermal component and usesthe patients own cultured keratinocytes; Integra™, which uses acollagen-glycosaminoglycan (GAG) matrix to provide an acellular dermalcomponent and uses a thin autograft; AlloDerm™ and a thin autograft;DermaGraft™, which uses a polyglycolic acid/polylactic acid (PGA/PLA)matrix and allogeneic human fibroblasts for the dermis;Hyaff/LaserSkin™, which uses hyaluran and fibroblasts for the dermis,and hyaluran and the patients own keratinocytes for the epidermis; andPolyActive™, which uses polyethylene oxide/polybutylthalate (PEO/PBT)and may use the patient's own fibroblasts for the dermis, and thepatient's cultured keratinocytes for the epidermis.

Materials to either temporarily cover wounds, or to stimulate permanentskin repair processes, include ApliGraft™, which uses collagen gel andallogeneic fibroblasts for the dermis, and cultured allogeneickeratinocytes for the epidermis; Comp Cult Skin™ or Orcel™ which usescollagen and allogeneic fibroblasts for the dermis, and culturedallogeneic keratinocytes for the epidermis; and TransCyte™ fibroblastsfor the dermis and a synthetic material, BioBrane™, for the epidermis.

While the above materials are useful to varying degrees, each hasdisadvantages and limitations. Some of the materials are fragilemechanically, making it difficult to perform the required manipulationsand transfers of the material in large sections without tearing.Instead, the materials must be used as smaller pieces, which makescoverage of large surface areas technically laborious for the physicianand cosmetically undesirable for the patient due to scarring wheregrafts adjoin. The materials are also susceptible to microbialcontamination, which is unacceptable for patients who are already at anincreased risk for infection due to their compromised conditions. Thematerials show varying rates of engraftment and times to heal, both ofwhich must be considered in selecting the advantages of a particularmaterial over another for a particular patient. For example, a materialwhich is otherwise acceptable but which takes longer to engraft and healis less desirable, because a successful recovery includes as rapid areturn to a normal routine as possible.

The inventor's own previous composite skin replacement, disclosed inU.S. Pat. No. 5,976,878, which is expressly incorporated by referenceherein in its entirety, had been successfully used for therapeutictreatment of skin wounds. It was applied surgically in a singleprocedure, and contained a layer of cultured epidermal cells, anacellular polymeric dermal membrane component, and a substantiallynonporous lamination layer on one surface of the dermal membranecomponent. The dermal membrane component was formed from collagen, orcollagen and a mucopolysaccharide compound, and was laminated with thesame collagen-, or collagen and mucopolysaccharide-containing solutionwith a volatile cryoprotectant. The substantially nonporous laminationlayer may be located between the dermal component and the layer ofcultured epidermal cells, promoting localization of epidermal cells onthe surface of the dermal component and movement of nutrients to thecells of the cellular epidermal component. This composition can also beused to deliver biologically active molecules to the site where it isapplied.

Desirable features of the above-described composite skin replacementincluded a more rapid rate of vascularization of the area covered by thematerial, decreased microbial contamination, increased nutrient supply,and improved epidermal barrier function, compared to other materials.Areas covered with the composite skin replacement required less time toengraft and heal, and the material was less susceptible to microbialcontamination than reported for other materials. Other desirablefeatures are that this material was relatively non-fragile and easy tohandle, and could be generated relatively rapidly, for example, withinthe time frame in which a burn patient requires skin grafts. However,while no other alternative material has healed excised, full-thicknesswounds more rapidly, and with as low an incidence of microbialcontamination, limitations still exist. Thus, there remains a need tomore closely approach structural and functional properties of normaluninjured skin.

SUMMARY OF THE INVENTION

The invention is directed to a device for surgical grafting of skinwounds, or for a model of skin in vitro and in animals. The device hasan acellular, biocompatible, reticulated protein- orpolypeptide-containing matrix to provide an attachment substrate for oneor more layers or populations of cultured dermal and/or epidermal cells.The protein can be naturally occurring or synthetic and may be less thana full protein, for example, it may be a polypeptide. In variousembodiments, cells used to populate the matrix may be from the recipient(autologous), another human (allogeneic), from another species(xenogeneic), or from multiple sources (chimeric). The epidermal cellsinclude keratinocytes, melanocytes, immunocytes, and/or stem cells. Thedermal cells include fibroblasts, endothelial cells, immunocytes, nervecells, myocytes, and/or stem cells. Either or both of the epidermal anddermal cells may be genetically modified.

The invention is also directed to a method to prepare a device forsurgical grafting of skin wounds, or for a model of skin in vitro and inanimals. To a matrix on an absorbent substrate, a cell suspension isprovided to deliver cells to the matrix for attachment. The inoculatedmatrix is incubated under conditions sufficient to result in a cellulardevice.

The device may also be used on a non-wounded surface, a minimallywounded surface, or a surgically prepared surface not requiring skingrafting, but for which the cells of the skin substitute may bebioengineered to provide a physiologic factor lacking in the recipient.Such a factor may be a protein, for example, insulin or coagulationFactor VIII, and may be provided, respectively, to a diabetic orhemophiliac patient having a deficiency of that protein.

Besides its use as a skin substitute, the inventive device can also beused as a living substrate on which to perform toxicological or othertests on various topically applied compounds, such as drugs, cosmetics,moisturizers, lotions, environmental toxins, industrial chemicals, etc.The device, containing cells from a particular individual, can show anindividualized response to a variety of compounds. Such an approach maybe useful to test the toxicity of skin contact compounds. The device mayalso be useful as a medical diagnostic tool to test individuals withallergies, or who exhibit dermal reactions to components found inpharmaceutical or over-the-counter products. In this embodiment, thedevice will reduce or eliminate in vivo toxicity testing.

These and other features of the invention will be appreciated withreference to the following detailed description.

DETAILED DESCRIPTION

The inventive, surgically-applied device for treatment of skin wounds isa matrix which supports dermal cells and/or epidermal cells. Moreparticularly, an acellular biocompatible reticulated matrix is used as asupport or scaffold to which cultured cells are applied, attach, andproliferate. In one embodiment, a reticulated protein matrix supports acontinuous layer or population of cultured dermal cells, and anoverlying layer or population of cultured epidermal cells. Afterincubating the inoculated matrix under conditions facilitating cellgrowth, the device is transplanted surgically to the patient. In oneembodiment, transplantation may be performed within one day (about 16hours to about 24 hours) after epidermal cell inoculation of the matrix.In another embodiment, transplantation may be performed within one monthafter epidermal cell inoculation of the matrix. Within these times, thedevice develops properties preferred for a therapeutic skin graftmaterial. The use of cultured cells to form the material, in contrast totissue obtained by conventional harvesting of split thickness skin witha dermatome, provides the advantage of much larger numbers of epidermaland dermal cells than by conventional harvesting, and thereby greatlyreduces the requirement for donor skin to complete closure of extensive,full-thickness skin wounds.

Once the device is grafted to the patient, the biodegradable matrix isabsorbed by the body. The cells organize to form functional skin tissue,referred to as an engrafted cultured skin substitute. The device hasmany of the properties and structures that are found in normal,uninjured skin, and functions as does normal, uninjured skin to protectthe individual from fluid loss and microbial infection. For example, thedevice functions as an epidermal barrier, which is definitive of normalskin function as known to those skilled in the art. The deviceestablishes a basement membrane, and maintains the same anatomicconfiguration of the cellular layers or populations as in normal,uninjured skin. The device produces and releases angiogenic factors andmediators of the inflammatory process, as does normal, uninjured skin.The device is effectively vascularized in less than one week, andbecomes partially vascularized within two days after transplant.

In another embodiment of the invention, the device is used as atemporary skin substitute. In this embodiment, the matrix may bepopulated with cells having non-autologous genotypes. For example,cultured epidermal and/or dermal cells may be autologous, that is,obtained from the individual who is the intended recipient of the deviceand which can be used in a permanently engrafted device. In otherembodiments, the epidermal and/or dermal cells may be allogeneic, thatis, obtained from a human other than the recipient. In yet otherembodiments, the epidermal and/or dermal cells may be xenogeneic, andobtained from a non-human animal, such as porcine epidermal and/ordermal cells to take advantage of the similarity of features andcharacteristics in pig skin in comparison to human skin. Xenogeneiccells may also be obtained from plants or microbes. The use of differentsources for epidermal cells and/or dermal cells results in a geneticallychimeric device. Regardless of the source of epidermal and/or dermalcells, one or more cells may be modified genetically. Various factorsmay affect the selection of particular genotypic compositions of thecells. For example, the use of allogeneic or xenogeneic cells mayshorten the preparation time of the device, or may further reduce therequirement for donor skin from the patient. Depending upon theparticular condition of the recipient, these factors may be an importantdeterminant.

If skin cells from the patient to be treated with the inventive deviceare used, they are obtained from a biopsy of a healthy area of thepatient's skin, using techniques known to one skilled in the artincluding punch biopsy, shave biopsy, and full thickness skin excisionwith suture closure. The dermal and epidermal cellular components arethen separated and isolated into dermal cells and epidermal cells, asdescribed by Boyce and Ham in J. Tissue Culture Methods 1985;9:83, andchapter 13 in In Vitro Models for Cancer Research, Vol. 3, p. 245,Webber and Sekely, Eds. CRC Press, Boca Raton Fla. (1986), both of whichare expressly incorporated by reference herein. The dermal and epidermalcells are individually cultured, as described by Boyce and Ham in J.Invest. Dermatol. 1983;81:335, and chapter 28 in Methods in MolecularMedicine, Vol. 18, p. 365, Morgan & Yarmush, Eds., Humana Press, TotowaN.J. (1998), both of which are expressly incorporated by referenceherein.

Various cells in the epidermis, for example, keratinocytes, melanocytes,immunocytes, stem cells, or others, and various cells in the dermis, forexample, fibroblasts, endothelial cells, immunocytes, nerve cells,myocytes, stem cells, or others, may be cultured either individually orcollectively. After adequate cell numbers are obtained, or a specificcellular physiology is expressed, the cellular populations are harvestedfor subsequent population of the matrix. In various embodiments, theratio of epidermal to dermal cells used to inoculate the matrix is inthe range of about 2:1 or 1:1, but other cell ratios are also included.

Depending upon the application for which the device is prepared,selected types of epidermal cells and/or dermal cells may be included orexcluded. As one example, a device may include melanocytes to restorepigmentation in the transplant site, Restoration of skin pigmentation isdefined as any increase in the anatomic or physiologic function of skincolor of the graft, although the extent of color may be more or lessthan in uninjured skin. As another example, a cultured skin compositionmay include endothelial cells to stimulate formation of blood vessels.

In preparing the device, any biocompatible material that is permissiveas a substrate for culture and transplantation of cultured cells may beused. A full length natural or synthetic protein may be used, or apolypeptide may be used. One embodiment uses a freeze-dried sponge ofcollagen, either alone or in combination with a carbohydrate (amucopolysaccharide, such as a glycosaminoglycan (GAG), particularlychondroitin-6-sulfate). The collagen may be bovine skin collagen, bovinetendon collagen, collagen from other tissue sources (e.g. bone, muscle),other xenogeneic sources (e.g. pig, sheep, goat, etc.), geneticallyengineered sources, human sources, or a combination of any of the above.Other proteins such as elastin or reticulin, or polymers of amino acids,whether naturally occurring or synthetic, may be used.

In one embodiment of preparing the matrix, a coprecipitate ofcollagen-GAG is cast, frozen, and dehydrated to form a reticulatedmatrix. This matrix is subsequently sterilized, rehydrated, andlaminated by inoculation with cultured dermal and epidermal cells.Inoculation is performed at ambient humidity (room air) and theinoculated matrix is incubated in an atmosphere with saturated orreduced humidity. The matrix is then incubated, either submerged in amedium or with the matrix contacting a gaseous atmosphere. In the latterembodiment, the inoculated cells are on the atmospheric surface of thematrix. Each of these steps is now described in further detail.

Matrix-Forming Protein-Containing Fluid

A dispersion of collagen is prepared by presolublizing collagen (6.42mg/ml) acetic acid (0.01 M to 1.0 M), usually for up to sixteen hours,after which the dispersion is stored at 4° C. A coprecipitate with aglycosaminoglycan (GAG), such as chondroitin-6-sulfate, may then beprepared if a carbohydrate is to be added. Chondroitin-6 sulfate (3.45mg/ml) is added to acetic acid (0.01 M to 3.0 M).

The previously prepared collagen dispersion is redispersed for at leastfive minutes and transferred to a stainless steel insulated beaker witha recirculating refrigerated jacket. The GAG solution is added to theprotein solution by any means which will produce an adequate agitationand shear to form a co-precipitate. This can be done by transferring theGAG solution to a drip bottle and adding the GAG to the collagen using adrip set to which a 22 gauge needle is attached, allowing the GAGsolution to drip into 750 ml of the collagen dispersion, being mixed ata speed of 5,000 revolutions per minute (rpm) and maintained at 4° C.,at a rate of one drop per ten seconds. After the entire volume of GAGhas dripped into the collagen, the collagen-GAG coprecipitate istransferred to bottles and centrifuged to remove trapped air bubbles.The froth that collects on top is removed by aspiration, and thecollagen-GAG coprecipitate is then collected.

Preparation of Crosslinked Matrix

The protein-containing fluid, with or without carbohydrate, is preparedto form the matrix. As preliminary steps, a lyophilizer (freeze-drying)apparatus is pre-chilled to about −35° C. to about −50° C. In oneembodiment, a freezing bath is prepared in a high density polyethylene(HDPE) container containing 95% ethanol that has been pre-chilled atabout −45° C. for at least four hours. However, any type of apparatus orconfiguration may be used which will remove heat at a controlled rate sothat a drop in temperature, sufficient to freeze the matrix, occurswithin a time frame of up to about four hours. For example, the time andtemperature may be regulated to bring about a temperature drop fromabout 4° C. to about −40° C. within about two hours, or a temperaturedrop from about 4° C. to about −75° C. within about four hours.

The protein solution is introduced into an apparatus, more fullydescribed in U.S. patent application Ser. No. 10/091,849, now U.S. Pat.No. 6,905,10 entitled “Apparatus for Preparing a Biocompatible Matrix”filed on Mar. 6, 2002, which is expressly incorporated by referenceherein in its entirety. The result is a matrix with a composition,structure, and properties which support the cultured dermal andepidermal cells to promote formation of the device.

Briefly, a matrix-forming solution is contained between two plates of athermally conductive material, with a gasket forming the remaining sidesof a sealed chamber. The thickness of the gasket, in the range of about0.1 mm to about 10 mm, regulates the thickness of the resulting matrix.The protein solution is introduced into the chamber. When the entirevolume of solution has been added, the chamber is reversibly sealed, forexample, by clamping. The chamber is then exposed to temperatures and/orconditions sufficient to remove heat at the previously-described,controlled rate to solidify the matrix.

After the matrix has solidified, the plates are separated to expose thefrozen matrix. A plate containing the matrix is transferred to arefrigerated (−45° C.) shelf of a lyophilizer. Vacuum is then appliedand, when the pressure is less than 60 mT, heat is also applied (30°C.). Lyophilization occurs overnight to a final vacuum of less than 15mT. The freeze-dried matrix detaches spontaneously and is thentransferred to a supporting sheet.

The matrix is cross-linked in the absence of a chemical crosslinkingagent. This desirably eliminates any possible toxicity associated withresidual chemical crosslinking agents, which may not be completelyremoved even after repeated washings. In one embodiment of theinvention, thermal crosslinking is used. This is achieved by thermaldehydration in a vacuum oven (Lab-Line 3628) at about −100 kPa at about105° C. for about 24 hours. Once crosslinking has occurred, the matrixis then stored in a desiccator at room temperature, either on a foilsheet or on other support material, for up to about three months.

The crosslinked matrix has a thickness of three millimeters or less. Invarious embodiments, and depending upon other factors such as a desiredsite of implantation, the crosslinked matrix has a thickness in therange of about 0.1 mm to about 1.0 mm, about 0.1 mm to about 2.0 mm, orabout 0.1 mm to about 3.0 mm. A matrix having a thickness in the rangeof about 0.1 mm to about 1.0 mm, when inoculated with cells asdescribed, results in a device having a thickness in the range of about50 μm to about 500 μm. When such a device is used to treat skin wounds,this thickness desirably promotes rapid vascularization, nutrientdelivery, population of the device with cells, and waste removal, anddesirably facilitates degradation of the matrix after transplant,leaving only the cellular components of the composition remaining.

The cross-linked matrix is then cut into desired sizes and/or shapes. Inone embodiment, it is cut into squares (for example, 9 cm×9 cm, 11 cm×11cm, or about 19 cm×19 cm) using a straight edge and scissors. The matrixis packaged in a sterilization pouch (for example, Self-Seal™), andstored at room temperature in a desiccator for up to about three months.

The matrix is sterilized before inoculation, for example, by gammairradiation at a dose of at least about 2.5 MRad (for example,SteriGenics, Westerville Ohio). Once sterilized, the matrixsterilization pouch is stored at room temperature in a desiccator for upto about one year.

Cellular Inoculation of the Matrix

All solutions are sterile filtered through a 0.22 μm filter, and allprocedures are performed using aseptic techniques, as known to oneskilled in the art.

The matrix is transferred to a container of any shape that will hold avolume of about 250 μl/cm² of matrix/incubation. The matrix is rinsedthree times, for thirty minutes each rinse, with Hepes-buffered saline(HBS) solution, and two times for thirty minutes each with Dulbecco'sModified Eagle's medium (DMEM) solution or other suitable solution, asknown to one skilled in the art.

After the final rinse, the medium is aspirated from the container and aninoculation frame is placed over the surface of the matrix. Theinoculation frame is a square or rectangular frame made from a materialthat is chemically unreactive (e.g., stainless steel, Teflon™), underphysiologic conditions (i.e., 37° C., saturated humidity, neutral pH,isotonic solutions). The frame is sufficiently massive (e.g., severalounces) to generate a seal to the movement of cells that are inoculatedwithin its perimeter. The seal may be increased by addition of a bevelon the side contacting the matrix to increase the mass/area ratio, butwith a sufficient amount of flat or rounded surface contacting thematrix to prevent cutting of the matrix. About 10-12 ml of supplementedDMEM, as will be described, is placed into the frame. The matrix andframe, containing supplemented DMEM, are permitted to equilibrate at 37°C./5% CO₂ for at least fifteen minutes before inoculating the matrixwith cells.

Cells may be inoculated either submerged or emerged into the rehydratedmatrix. In one embodiment, termed “submerged inoculation”, cells areinoculated on a matrix submerged in medium. Culture medium without cellsis added to the culture vessel outside of the inoculation frame toassure a secure seal, evidenced by no leakage of the medium from outsideto inside the frame. After the preparation of a cell suspension bytrypsinization of cells from selective cultures, dermal cells areinoculated at a density in the range of about 0.05−1.0×10⁶ cells/cm².Subsequently, after the dermal cells have attached, epidermal cells areinoculated as suspensions and permitted to attach to the layer orpopulation of dermal cells. Alternatively, combinations of dermal andepidermal cells may be inoculated simultaneously. The ratio of dermalcells to epidermal cells may be in the range of about 2:1 to about 1:1,but other ratios may be used. In other embodiments, dermal cells aloneor epidermal cells alone may be inoculated.

The inoculation frame remains in place for about 12-48 hours afterinoculation of the last cells onto the matrix. The inoculation frame isthen removed, the edges of the matrix without cells are excised, and theinoculated surface of the matrix is exposed to the air to stimulateorganization of the epidermal cells and the formation of an epidermalbarrier. Before removing the inoculation frame, Dulbecco's ModifiedEagle's medium with permissive supplements is used. After removing theframe and exposing the matrix to air, the medium is supplemented withprogesterone and epidermal growth factor.

In another embodiment, termed “lifted inoculation”, cells are inoculatedon a matrix emerged from the culture medium. In this embodiment, thematrix is rehydrated and placed onto an absorbent substrate, with theupper surface contacting the atmosphere. The suspension of dermal cellsis inoculated onto the matrix, and the drainage of the medium deliversthe cells to the surface of the matrix, after which they attach.Simultaneously, or after up to one week, a suspension of epidermal cellsis inoculated onto the matrix.

More specifically, a sterile, non-adherent, porous membrane (e.g.,medical grade mesh (N-Terface7, Winfield Laboratories, Inc., DallasTex.); Teflon™; Millipore or Whatman filters of polyethersulfone,polyvinylidene fluoride, mixed cellulose ester, etc., hereinafterreferred to as a porous membrane) is placed into a sterile tissueculture dish with HBS, and the sterile matrix is placed on top of theporous membrane and rehydrated. A sterile, absorbent material (e.g.,Merocel™ that is 9 mm thick and of intermediate density (CF 100);cotton, gauze, etc., hereinafter referred to as an absorbent material)is placed into a second sterile dish to which excess DMEM is added. Thedish is returned to the incubator to equilibrate.

Preparatory to inoculating dermal cells, the matrix is centered on theporous membrane and the medium is aspirated. The matrix/porous membraneis laid on top of the absorbent material. The area of the matrix ismeasured to the nearest 0.5 cm and the dish is reincubated. Dermal cellsare harvested and counted. The density is adjusted to 3×10⁶ cells/mlwith supplemented DMEM, and about 5×10⁵ dermal cells/cm² are inoculatedonto the matrix. Supplemented DMEM is added, and the dish is returned tothe incubator.

On the following day, the unit is transferred to a sterile 150 mm dishcontaining 25 ml of supplemental DMEM containing progesterone andepidermal growth factor, hereinafter referred to as UCMC 160.The mediumis aspirated and an additional 25 ml of fresh UCMC 160 medium is added.The process is repeated daily until inoculation of epidermal cells.

Preparatory to inoculation of epidermal cells, sterile absorbentmaterial is placed in a sterile dish saturated with UCMC 160 medium andincubated. Several hours prior to the inoculation, the previouslyinoculated cell/matrix/porous membrane unit is placed on top of theabsorbent material. The area of the matrix is measured to the nearest0.5 cm, and the dish is reincubated. Epidermal cells are harvested andcounted. The density is adjusted to 1.2×10⁷ cells/mi UCMC 160 medium,and the matrix is inoculated with 1×10⁶ cells/cm², using the tip of thepipette to break the surface tension of the inoculum and make acontinuous layer of epidermal cells on the inoculated matrix. After30-60 minutes of incubation, UCMC 160 medium is added to the outside ofthe absorbent material. The inoculated matrix is incubated (day 0).

On day 1, the medium around the absorbent material is aspirated andfresh medium is added before reincubation. On day 2, a sterile liftingframe, consisting of wire mesh and cotton, is placed into a new steriledish and the appropriate volume of UCMC 160 medium is added to bring themedium into contact with the wire mesh and cotton. The inoculated matrixis moved onto the lifting frame and saturated cotton, and isreincubated. The process is repeated on day 3. From day 4 onward, theprocess is repeated using supplemented UCMC 161 medium.

UCMC 161 medium is used for the inoculated matrix. To a base of DMEMwith reduced phenol red, the following supplements (all available fromSigma, St. Louis Mo.) are added to achieve a final concentration withinthe ranges as indicated: strontium chloride (0.01 mM to 100 mM);linoleic acid/BSA (0.02 μg/ml to 200 μg/ml); insulin (0.05 μg/ml to 500μg/ml); triiodothyronine (0.2 pM to 2000 pM); hydrocortisone (0.005 82μg/ml to 50 μg/ml); a combination of penicillin (100 U/ml), streptomycin(100 μg/ml), amphotericin (0.25 μg/ml); and ascorbic acid-2-phosphate(0.001 mM to 10 mM).

To prepare UCMC 160 medium, progesterone (0.1 nM to 1000 nM) andepidermal growth factor (0.01 ng/ml to 100 ng/ml) are added to UCMC 161medium to promote transient proliferation of keratinocytes.

Without being bound by a specific theory or mechanism, the followingevents likely occur. Upon inoculation, fibroblasts likely form aphysiological attachment to the collagen matrix by binding viacollagen-specific receptors. Because the matrix is reticulated and thuscontains multiple continuous surfaces, as opposed to being perforatedwith direct channels or openings from a top surface to a bottom surface,the fibroblasts or other dermal cells being inoculated need not fillthese channels or openings in the matrix before the epidermal cells maybe added. Rather, upon inoculation, the dermal cells attach to thereticulations, and thus are able to provide a continuous surfacelamination for the subsequently inoculation of epidermal cells within ashorter time period than is possible using a perforated matrix.

After inoculation, the device is incubated under conditions facilitatingcell growth, maintenance, and division anywhere from less than one day(within about 16 hours to about 24 hours) up to about six weeks. Thecells form a substantially continuous monolayer or multilayer surface.The device may then be transplanted into a patient, or it may beretained under these conditions until transplant. During this period,the matrix desirably degrades, cells proliferate, and new human collagenand biopolymers are deposited, all of which promote vascularization andengraftment of the device.

Engraftment of the Device

Preparatory to surgical transplantation of the device, the wound isprepared by minimizing microbial contamination and maximizing vascularsupply. These conditions are usually accomplished by early (i.e., lessthan one week post burn) tangential excision of burn eschar to a viablebase, and temporary protection of the excised wound with cadaverallograft skin or with a dermal substitute (i.e., Integra ArtificialSkin®).

At the time of transplantation, the temporary component of the allograftor dermal substitute is removed to generate a highly viable graft bedwith low microbial contamination. Hemostasis is attained, and one ormore of the cultured skin devices are transplanted and attached withsurgical staples. The device is dressed with non-adherent dressing(e.g., N-Terface®), fine-meshed cotton gauze, and bulky cotton gauze,with perforated catheters for irrigation of the device, for example,with a solution containing non-cytotoxic antimicrobial agents. Dressingchanges and examination are performed on postoperative days 2 and 5,after which time the wet dressings are typically discontinued, and anappropriate antimicrobial ointment (for example, equal partsNeomycin:Bactoban:Nystatin) is applied. The ointment is applied tounhealed areas until healing is complete. Once engrafted, various agentsthat may facilitate the healing process and/or minimize potentialcomplications may be applied topically to the device. For example, anutrient solution such as a modified cell culture medium can supplynutrients to the wound during vascularization, and/or a non-cytotoxicantimicrobial solution can reduce or control microbial contamination.

The inventive device may also be used for in vitro testing. For example,the device may be used for the evaluation of compounds intended forapplication to the skin, such as cosmetics and/or topical therapeutic orpreventative agents, or may be used for the evaluation of compoundswhich may contact the skin inadvertently, such as industrial chemicalsand/or environmental toxins. Information derived using the inventivedevice for any of these agents will be beneficial in a variety ofapplications. As one example, it may allow determination of a singleagent's, or a combination of agents', absorption, distribution,biotransformation, and elimination parameters in skin. As anotherexample, it may allow determination of a single agent's, or acombination of agents', toxicity to one or more cell types in skin. Asyet another example, it may allow qualitative and quantitativeassessment of a single agent's, or a combination of agent's, uptake inskin for formulation, permeability, and dosimetry studies. As stillanother example, it may allow evaluation of barrier function upon insultby a single agent or a combination of agents. Other examples ofapplications will be appreciated by one skilled in the art. Such methodshave a variety of benefits: they reduce or eliminate the need to conductin vivo studies, they allow more controlled screening comparisons andhence provide more reproducible data, they permit administration ofotherwise toxic chemicals and/or radiolabeled agents, etc. Additionally,the above-described and similar assessments may be customized by usingcells from a particular individual, for example, an individual prone toallergic reactions.

Methods of using the device for in vitro testing involve, generally,preparing the device or using a prepared device, and applying the agentto the device. The agent may be applied, either directly or indirectly,to any surface of the device, and/or may be added to the medium in whichthe device is incubated, and/or may be added within an environmentsurround the device, etc. The agent may also be inoculated into thedevice.

A cultured skin device and method of preparing the device is thusdisclosed. The inventive device and method provide treatment of skinwounds, and have structural and functional characteristics of normaluninjured skin. In one embodiment, the device contains cells from thepatient to whom it is applied, thus reducing or eliminating the concernof donor compatibility. Other variations or embodiments of the inventionwill also be apparent to one of ordinary skill in the art from the abovedescription. As one example, cells from non-human animals may be used toproduce a device for veterinary applications. As another example, thebiocompatible reticulated matrix may be acellular, or may contain only adermal cell component, or only an epidermal cell component. As yet otherexamples, the epidermal cells may be only melanocytes, or the dermalcells may be only endothelial cells. Thus, the forgoing embodiments arenot to be construed as limiting the scope of this invention.

What is claimed is:
 1. A method to provide a physiological factor in apatient in need thereof, the method comprising, surgically preparing askin surface to receive a skin substitute, then providing to theprepared surface a skin substitute comprising cultured dermal cellsdirectly attached to a biocompatible reticulated acellular matrix, thematrix prepared from a matrix-forming collagen-containing fluid that iscast, frozen, and dehydrated, the dermal cells providing a cellularlamination layer with cultured epidermal cells directly inoculated anddirectly attached thereon which establishes a basement membrane betweenthe epidermal cells and the dermal cells, at least one of the dermal orepidermal cells bioengineered to provide a physiologic factor in thepatient.
 2. The method of claim 1 wherein the physiologic factor is aprotein.
 3. The method of claim 1 wherein the physiologic factor isinsulin.
 4. The method of claim 1 wherein the physiologic factor isFactor VIII.
 5. The method of claim 1 wherein the physiologic factor isan angiogenic factor.
 6. The method of claim 1 wherein the physiologicfactor is an inflammatory process mediator.
 7. The method of claim 3wherein the patient is a diabetic.
 8. The method of claim 4 wherein thepatient is a hemophiliac.
 9. The method of claim 5 wherein the patientis in need of enhanced vascularization.
 10. A cultured skin devicecomprising cultured dermal cells, wherein at least one dermal cell isselected from the group consisting of autologous cells, allogeneiccells, xenogenic cells, chimeric cells, and combinations thereof,directly attached to a biocompatible reticulated acellular matrix, thematrix prepared from a matrix-forming collagen-containing fluid that iscast, frozen, and dehydrated, the dermal cells providing a cellularlamination layer with cultured epidermal cells, wherein at least oneepidermal cell is selected from the group consisting of autologouscells, allogeneic cells, xenogenic cells, chimeric cells, andcombinations thereof, directly inoculated and directly attached thereonwhich establishes a basement membrane between the epidermal cells andthe dermal cells.
 11. The device of claim 10 wherein all dermal cellsand all epidermal cells are from a single source.
 12. The device ofclaim 10 wherein all dermal cells are from a first source, and allepidermal cells are from a source other than the first source.
 13. Thedevice of claim 10 wherein all epidermal cells are from a first source,and all dermal cells are from a source other than the first source. 14.The device of claim 10 wherein all dermal cells and all epidermal cellsare autologous.
 15. The device of claim 10 wherein all dermal cells andall epidermal cells are allogeneic.
 16. The device of claim 10 whereinthe dermal or epidermal cells are autologous, and the non-autologouscells are allogeneic.
 17. The device of claim 10 wherein at least onedermal cell is autologous, and at least one epidermal cell is selectedfrom the group consisting of autologous, allogeneic, xenogenic, and/orchimeric.
 18. The device of claim 10 wherein at least one epidermal cellis autologous, and at least one dermal cell is selected from the groupconsisting of autologous, allogeneic, xenogenic, and/or chimeric. 19.The device of claim 10 wherein at least one cell is genetically modified20. A method of producing a cultured skin device, the method comprisingisolating at least a first dermal cell from a source selected from thegroup consisting of autologous, allogeneic, xenogeneic, and/or chimericdermal cells; culturing the isolated cells, and inoculating the culturedcells to a biocompatible reticulated acellular matrix, the matrixprepared from a matrix-forming collagen-containing fluid that is cast,frozen, and dehydrated, and incubating the inoculated matrix underconditions to form at least one dermal cellular lamination layerpopulation wherein a basement membrane is established between the dermalcells and epidermal cells directly inoculated and attached thereon, theepidermal cells from a source selected from the group consisting ofautologous, allogeneic, xenogeneic, and/or chimeric dermal cells. 21.The method of claim 20 wherein all dermal cells and all epidermal cellsare from a single source.
 22. The method of claim 20 wherein all dermalcells are from a first source, and all epidermal cells are from a sourceother than the first source.
 23. The method of claim 20 wherein allepidermal cells are from a first source, and all dermal cells are from asource other than the first source.
 24. The method of claim 20 whereinat least one dermal cell is autologous, and at least one epidermal cellis selected from the group consisting of autologous, allogeneic,xenogeneic, and/or chimeric cells.
 25. The method of claim 20 wherein atleast one epidermal cell is autologous, and at least one dermal cell isselected from the group consisting of autologous, allogeneic,xenogeneic, and/or chimeric cells.
 26. A method to decrease transplanttime and/or need for donor skin as a source of dermal or epidermalcells, the method comprising providing a device comprising cultureddermal cells directly attached to a biocompatible reticulated acellularmatrix, the matrix prepared from a matrix-forming collagen-containingfluid that is cast, frozen, and dehydrated, the dermal cells providing acellular lamination layer with cultured epidermal cells directlyinoculated and directly attached thereon which establishes a basementmembrane between the epidermal cells and the dermal cells, the dermaland/or epidermal cells supplemented at least in part with allogeneic,xenogeneic, and/or chimeric cells, the reduced donor dermal and/orepidermal cells resulting in a device available for transplant and/orrequiring reduced donor skin due to the allogeneic, xenogeneic, and/orchimeric cells.
 27. The method of claim 26 in veterinary applications.28. The method of claim 26 where the cells are xenogeneic and areselected from the group consisting of porcine cells, plant cells, andmicrobial cells.
 29. A pre-fabricated synthetic skin device comprisingcultured autologous and/or allogeneic dermal cells directly attached toa biocompatible reticulated acellular matrix in a sterile mediumcontaining progesterone and epidermal growth factor, the matrix preparedfrom a matrix-forming collagen-containing fluid that is cast, frozen,and dehydrated, the dermal cells providing a cellular lamination layerfor cultured epidermal cells capable of direct inoculation and directattachment thereon to establish a basement membrane between theepidermal cells and the dermal cells, the device stored under sterileconditions with a daily medium replacement until inoculation ofepidermal cells.
 30. The device of claim 29 subsequently inoculated withabout 1×10⁶ cells/cm² autologous or allogeneic epidermal cells andthereafter stored in a sterile medium up to six weeks.
 31. A method ofminimizing the number of skin grafts required to a patient in needthereof, the method comprising preparing a device using an apparatussized to simulate a graft site, the device prepared by inoculatingcultured dermal cells directly attached to a biocompatible reticulatedacellular matrix, the matrix prepared from a matrix-formingcollagen-containing fluid that is cast, frozen, and dehydrated, thedermal cells providing a cellular lamination layer with culturedepidermal cells directly inoculated and directly attached thereon whichestablishes a basement membrane between the epidermal cells and thedermal cells, resulting in a device covering a graft site in a recipient32. The method of claim 31 where the apparatus comprises a chambercontaining a matrix-forming fluid, the chamber defined by at least a topplanar rigid metal surface and a bottom planar rigid metal surface, saidtop and bottom surfaces effective to symmetrically remove heat from thematrix-forming fluid in preparation of the biocompatible matrix, atleast one discontinuous gasket having a uniform thickness positionedbetween said top and bottom surfaces to define a perimeter of saidchamber, said gasket capable of containing said matrix-forming fluidwithin said perimeter, and a plurality of fasteners to fasten said topsurface with said bottom surface.
 33. A method of inoculating abiocompatible reticulated matrix with enhanced engraftment, the methodcomprising inoculating fibroblasts on a biocompatible reticulatedcollagen matrix, the matrix prepared from a matrix-formingcollagen-containing fluid that is cast, frozen, and dehydrated, thefibroblasts binding to collagen-specific receptors forming aphysiological attachment to the collagen matrix by binding viacollagen-specific receptors forming a continuous surface laminationlayer on the matrix, and inoculating cultured epidermal cells directlyon the lamination layer in a shorter time than is possible if using aperforated matrix, the epidermal cells directly attached thereonestablishing a basement membrane between the epidermal cells and thedermal cells, resulting in enhanced engraftment.